Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster

Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies. By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture. The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug. We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own.At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper 5: Manuscrip

Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses